Hypofibrinogenemia and liver disease: a new case of Aguadilla fibrinogen and review of the literature.

INTRODUCTION Fibrinogen storage disease (FSD) is characterized by hypofibrinogenemia and hepatic inclusions due to impaired release of mutant fibrinogen which accumulates and aggregates in the hepatocellular endoplasmic reticulum. Liver disease is variable. AIM We studied a new Swiss family with fibrinogen Aguadilla. In order to understand the molecular peculiarity of FSD mutations, fibrinogen Aguadilla and the three other causative mutations, all located in the γD domain, were modelled. METHOD The proband is a Swiss girl aged 4 investigated because of fatigue and elevated liver enzymes. Protein structure models were prepared using the Swiss-PdbViewer and POV-Ray software. RESULTS The proband was found to be heterozygous for fibrinogen Aguadilla: FGG Arg375Trp. Familial screening revealed that her mother and maternal grandmother were also affected and, in addition, respectively heterozygous and homozygous for the hereditary haemochromatosis mutation HFE C282Y. Models of backbone and side-chain interactions for fibrinogen Aguadilla in a 10-angstrom region revealed the loss of five H-bonds and the gain of one H-bond between structurally important amino acids. The structure predicted for fibrinogen Angers showed a novel helical structure in place of hole 'a' on the outer edge of γD likely to have a negative impact on fibrinogen assembly and secretion. CONCLUSION The mechanism by which FSD mutations generate hepatic intracellular inclusions is still not clearly established although the promotion of aberrant intermolecular strand insertions is emerging as a likely cause. Reporting new cases is essential in the light of novel opportunities of treatment offered by increasing knowledge of the degradation pathway and autophagy.

Combined effect of 25-OH vitamin D plasma levels and genetic vitamin D receptor (NR 1I1) variants on fibrosis progression rate in HCV patients

Decreased vitamin D levels have been described in various forms of chronic liver disease and associated with advanced fibrosis. Whether this association is a cause or consequence of advanced fibrosis remains unclear to date.

Extracts of Lindera obtusiloba induce antifibrotic effects in hepatic stellate cells via suppression of a TGF-beta-mediated profibrotic gene expression pattern

Liver fibrosis is characterized by high expression of the key profibrogenic cytokine transforming growth factor (TGF)-beta and the natural tissue inhibitor of metalloproteinases (TIMP)-1, leading to substantial accumulation of extracellular matrix. Liver fibrosis originates from various chronic liver diseases, such as chronic viral hepatitis that, to date, cannot be treated sufficiently. Thus, novel therapeutics, for example, those derived from Oriental medicine, have gained growing attention. In Korea, extracts prepared from Lindera obtusiloba are used for centuries for treatment of inflammation, improvement of blood circulation and prevention of liver damage, but experimental evidence of their efficacy is lacking. We studied direct antifibrotic effects in activated hepatic stellate cells (HSCs), the main target cell in the fibrotic liver. L. obtusiloba extract (135 mug/ml) reduced the de novo DNA synthesis of activated rat and human HSCs by about 90%, which was not accompanied by cytotoxicity of HSC, primary hepatocytes and HepG2 cells, pointing to induction of cellular quiescence. As determined by quantitative polymerase chain reaction, simultaneous treatment of HSCs with TGF-beta and L. obtusiloba extract resulted in reduction of TIMP-1 expression to baseline level, disruption of the autocrine loop of TGF-beta autoinduction and increased expression of fibrolytic matrix metalloproteinase (MMP)-3. In addition, L. obtusiloba reduced gelatinolytic activity of HSC by interfering with profibrogenic MMP-2 activity. Since L. obtusiloba extract prevented intracellular oxidative stress experimentally induced by tert-butylhydroperoxide, we concluded that the direct antifibrotic effect of L. obtusiloba extract might be mediated by antioxidant activity. Thus, L. obtusiloba, traditionally used in Oriental medicine, may complement treatment of chronic liver disease.

Factors affecting screening for hepatocellular carcinoma

Background and aim. Hepatocellular carcinoma (HCC) is a frequent cancer. Its prognosis is highly dependent on early diagnosis. Patients at risk for developing HCC should be enrolled in a surveillance programme. Nevertheless, many patients at risk are not regularly screened. We aimed at exploring the characteristics that affect enrolment in a surveillance programme. Material and methods. The characteristics of the patients included in the prospective Bern HCC cohort between August 2010 and August 2011 were analysed according to their participation in a surveillance programme. Results. Among the 82 patients included in the cohort during this period of time, 48 were in a surveillance program before the diagnosis of HCC. Thirty five percent of cirrhotic patients were not screened. Age, sex, level of education, Child-Pugh status and MELD score were similar between the patients who were screened and those who were not screened. Patients with a private insurance and patients treated by a liver specialist were more frequently enrolled in a surveillance program. Sixty seven percent of the screened patients were eligible for curative treatment whereas only 15% of the non-screened patients were. Conclusions. In conclusion the surveillance of patients at risk for developing HCC increases their chances to be diagnosed at an early stage to allow curative treatment. More than one third of cirrhotic patients were not regularly screened. Patients with chronic liver disease should be referred to identify those at risk and enrol them in a surveillance program.

Vascular cell adhesion molecule 1 expression by biliary epithelium promotes persistence of inflammation by inhibiting effector T-cell apoptosis

Chronic hepatitis occurs when effector lymphocytes are recruited to the liver from blood and retained in tissue to interact with target cells, such as hepatocytes or bile ducts (BDs). Vascular cell adhesion molecule 1 (VCAM-1; CD106), a member of the immunoglobulin superfamily, supports leukocyte adhesion by binding a4b1 integrins and is critical for the recruitment of monocytes and lymphocytes during inflammation. We detected VCAM-1 on cholangiocytes in chronic liver disease (CLD) and hypothesized that biliary expression of VCAM-1 contributes to the persistence of liver inflammation. Hence, in this study, we examined whether cholangiocyte expression of VCAM-1 promotes the survival of intrahepatic a4b1 expressing effector T cells. We examined interactions between primary human cholangiocytes and isolated intrahepatic T cells ex vivo and in vivo using the Ova-bil antigen-driven murine model of biliary inflammation. VCAM-1 was detected on BDs in CLDs (primary biliary cirrhosis, primary sclerosing cholangitis, alcoholic liver disease, and chronic hepatitis C), and human cholangiocytes expressed VCAM-1 in response to tumor necrosis factor alpha alone or in combination with CD40L or interleukin-17. Liver-derived T cells adhered to cholangiocytes in vitro by a4b1, which resulted in signaling through nuclear factor kappa B p65, protein kinase B1, and p38 mitogen-activated protein kinase phosphorylation. This led to increased mitochondrial B-cell lymphoma 2 accumulation and decreased activation of caspase 3, causing increased cell survival. We confirmed our findings in a murine model of hepatobiliary inflammation where inhibition of VCAM-1 decreased liver inflammation by reducing lymphocyte recruitment and increasing CD8 and T helper 17 CD4 Tcell survival. Conclusions: VCAM-1 expression by cholangiocytes contributes to persistent inflammation by conferring a survival signal to a4b1 expressing proinflammatory T lymphocytes in CLD.

Reduced IFNλ4 activity is associated with improved HCV clearance and reduced expression of interferon-stimulated genes

Hepatitis C virus (HCV) infections are the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. Both spontaneous and treatment-induced clearance of HCV depend on genetic variation within the interferon-lambda locus, but until now no clear causal relationship has been established. Here we demonstrate that an amino-acid substitution in the IFNλ4 protein changing a proline at position 70 to a serine (P70S) substantially alters its antiviral activity. Patients harbouring the impaired IFNλ4-S70 variant display lower interferon-stimulated gene (ISG) expression levels, better treatment response rates and better spontaneous clearance rates, compared with patients coding for the fully active IFNλ4-P70 variant. Altogether, these data provide evidence supporting a role for the active IFNλ4 protein as the driver of high hepatic ISG expression as well as the cause of poor HCV clearance.

Results of the Questionnaire

Early diagnosis of patients with compensated advanced chronic liver disease (cACLD) and portal hypertension is challenging in clinical practice. A growing amount of evidence regarding noninvasive diagnostic methods, and in particular liver stiffness measurement (LSM), suggests that these tools could be used in clinical practice and might potentially limit the use of invasive, reference diagnostic tools (HVPG measurement and endoscopy). Our panel aimed at better understanding the opinion of the Baveno faculty regarding the current practice and use of invasive and noninvasive methods in the field of screening and surveillance of varices; a specific questionnaire was electronically sent to all the faculty members. The results suggested that the experts agreed on the use of noninvasive methods to rule out/identify patients with cACLD. They also indicated that the persistence or removal of the causal agent which led to cirrhosis should guide the choice of using the shortest or the longest interval among those recommended for surveillance endoscopies. Finally, the use of noninvasive methods in these clinical scenarios was pointed out as a relevant field for future research.

Consensus Statements: Session 1 - Screening and Surveillance

The introduction of transient elastography in clinical practice has allowed the early identification of patients with chronic liver disease (CLD) at risk of developing clinically significant portal hypertension (CSPH) (1b;A).

Cannabinoid receptor type I modulates alcohol-induced liver fibrosis

The cannabinoid system (CS) is implicated in the regulation of hepatic fibrosis, steatosis and inflammation, with cannabinoid receptors 1 and 2 (CB1 and CB2) being involved in regulation of pro- and antifibrogenic effects. Daily cannabis smoking is an independent risk factor for the progression of fibrosis in chronic hepatitis C and a mediator of experimental alcoholic steatosis. However, the role and function of CS in alcoholic liver fibrosis (ALF) is unknown so far. Thus, human liver samples from patients with alcoholic liver disease (ALD) were collected for analysis of CB1 expression. In vitro, hepatic stellate cells (HSC) underwent treatment with acetaldehyde, Δ9-tetrahydrocannabinol H(2)O(2), endo- and exocannabinoids (2-arachidonoylglycerol (2-AG) and [THC]), and CB1 antagonist SR141716 (rimonabant). In vivo, CB1 knockout (KO) mice received thioacetamide (TAA)/ethanol (EtOH) to induce fibrosis. As a result, in human ALD, CB1 expression was restricted to areas with advanced fibrosis only. In vitro, acetaldehyde, H(2)O(2), as well as 2-AG and THC, alone or in combination with acetaldehyde, induced CB1 mRNA expression, whereas CB1 blockage with SR141716 dose-dependently inhibited HSC proliferation and downregulated mRNA expression of fibrosis-mediated genes PCα1(I), TIMP-1 and MMP-13. This was paralleled by marked cytotoxicity of SR141716 at high doses (5-10 μmol/L). In vivo, CB1 knockout mice showed marked resistance to alcoholic liver fibrosis. In conclusion, CB1 expression is upregulated in human ALF, which is at least in part triggered by acetaldehyde (AA) and oxidative stress. Inhibition of CB1 by SR141716, or via genetic knock-out protects against alcoholic-induced fibrosis in vitro and in vivo.

Role of hepatitis C virus genotype 3 in liver fibrosis progression - a systematic review and meta-analysis

The progression of liver fibrosis in chronic hepatitis C has long been considered to be independent from viral genotypes. However, recent studies suggest an association between Hepatitis C virus (HCV) genotype 3 and accelerated liver disease progression. We completed a systematic review and meta-analysis of studies evaluating the association between HCV genotypes and fibrosis progression. PubMed, Embase and ISI Web of Knowledge databases were searched for cohort, cross-sectional and case-control studies on treatment-naïve HCV-infected adults in which liver fibrosis progression rate (FPR) was assessed by the ratio of fibrosis stage in one single biopsy to the duration of infection (single-biopsy studies) or from the change in fibrosis stage between two biopsies (paired biopsies studies). A random effect model was used to derive FPR among different HCV genotypes. Eight single-biopsy studies (3182 patients, mean/median duration of infection ranging from 9 to 21 years) and eight paired biopsies studies (mean interval between biopsies 2-12 years) met the selection criteria. The odds ratio for the association of genotype 3 with accelerated fibrosis progression was 1.52 (95% CI 1.12-2.07, P = 0.007) in single-biopsy studies and 1.37 (95% CI 0.87-2.17, P = 0.17) in paired biopsy studies. In conclusion, viral genotype 3 was associated with faster fibrosis progression in single-biopsy studies. This observation may have important consequences on the clinical management of genotype 3-infected patients. The association was not significant in paired biopsies studies, although the latter may be limited by important indication bias, short observation time and small sample size.

[Liver transplantation - when and for whom it should be performed]

During the past two decades, orthotopic liver transplantation (OLT) emerged to the treatment of choice for patients with end-stage liver disease. In Switzerland, about 100 liver transplantations are performed every year, while the shortage of cadaveric organs considerably outmatches the demand. Common indications for OLT include cirrhosis due to alcoholic liver disease or chronic viral hepatitis related to hepatitis B or C, and hepatocellular carcinoma. With the advent of the new allocation policy in Switzerland in 2007, patients listed for OLT are mainly stratified based on the Model of End-stage Liver Disease (MELD) score. Using a patient's laboratory values for serum bilirubin, serum creatinin, and the international normalized ratio for prothrombin time (INR), the MELD score accurately predicts three-month mortality among patients on the waiting list. Compared to the pre-MELD era, patients with significantly higher MELD scores undergo transplantation which leads in turn to more complications and higher costs yet with a comparable outcome. Timely referral of potential candidates to a transplant center is crucial since thorough evaluation to rule out contraindications such as uncontrolled infection, extrahepatic malignancy or advanced cardiopulmonary disease is essential. Taken together, every patient presenting with acute liver failure, decompensated cirrhosis or suspected hepatocellular carcinoma should be evaluated in a center with liver transplantation capability.

Valve disease in chronic venous disorders: a quantitative ultrastructural analysis by transmission electron microscopy and stereology

INTRODUCTION: The ultrastructure of venous valves and walls in chronic venous disease was investigated. METHODS: Consecutive patients were categorised into one of three groups (group A: patients with C1 venous disease in accordance with CEAP (Clinical severity, Etiology, Anatomy, Pathophysiology); group B: C2 and C3; group C: C4, C5 and C6). The terminal or preterminal valve and adjacent vessel wall was harvested from the great saphenous vein. Sections were examined with a transmission electron microscope. The volumes of elastin and of collagen per unit surface area of valve were assessed, as well as the surface endothelium of valve and vessel wall. RESULTS: The study population consisted of 17 patients. The elastin ratio was analysed by means of stereology. Mean values were: in group A, 0.45 μm3/m2; in group B, 0.67 μm3/m2; in group C, 0.97 μm3/m2. The ratio was similar for collagen (A, 15.7 μm3/m2; B, 26.8 μm3/m2; C, 30.1 μm3/m2). Surface analysis of the valve endothelium and the adjacent vessel wall endothelium showed a trend towards increasing damage with more severe disease. CONCLUSIONS: With progression of venous disease, the valve elastin content, assessed morphologically, seems to increase, and the endothelium of the venous valve and the vein wall tend to show more damage.

Non-coding keratin variants associate with liver fibrosis progression in patients with hemochromatosis

Background Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis. Methods The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed. Results We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury. Conclusion In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.

A common variant of PNPLA3 (p.I148M) is not associated with alcoholic chronic pancreatitis

Background Chronic pancreatitis (CP) is an inflammatory disease that in some patients leads to exocrine and endocrine dysfunction. In industrialized countries the most common aetiology is chronic alcohol abuse. Descriptions of associated genetic alterations in alcoholic CP are rare. However, a common PNPLA3 variant (p.I148M) is associated with the development of alcoholic liver cirrhosis (ALC). Since, alcoholic CP and ALC share the same aetiology PNPLA3 variant (p.I148M) possibly influences the development of alcoholic CP. Methods Using melting curve analysis we genotyped the variant in 1510 patients with pancreatitis or liver disease (961 German and Dutch alcoholic CP patients, 414 German patients with idiopathic or hereditary CP, and 135 patients with ALC). In addition, we included in total 2781 healthy controls in the study. Results The previously published overrepresentation of GG-genotype was replicated in our cohort of ALC (p-value <0.0001, OR 2.3, 95% CI 1.6–3.3). Distributions of genotype and allele frequencies of the p.I148M variant were comparable in patients with alcoholic CP, idiopathic and hereditary CP and in healthy controls. Conclusions The absence of an association of PNPLA3 p.I148M with alcoholic CP seems not to point to a common pathway in the development of alcoholic CP and alcoholic liver cirrhosis.

The vitamin D receptor gene bAt (CCA) haplotype impairs the response to pegylated-interferon/ribavirin-based therapy in chronic hepatitis C patients

Chronic hepatitis C infection is a major cause of end-stage liver disease. Therapy outcome is influenced by 25-OH vitamin D deficiency. To further address this observation, our study investigates the impact of the vitamin D receptor (NR1I1) haplotype and combined effects of plasma vitamin D levels in a well-described cohort of hepatitis C patients.